| As female gametes,oocytes arrested at the stage of prophase of first meiosis,which is also termed as germinal vesicle(GV)stage,through the time of birth till to puberty.Oocytes begin to resume meiosis and germinal vesicle breakdown(GVBD)only after puberty,which is stimulated by gonadotropin surge,and only after meiotic resumption can fully-grown oocytes be fertilized.In mammalian oocytes,a decline in cyclic adenosine monophosphate(cAMP),which brought by the changes of phosphodiesterases(PDEs),is thought to initiate GVBD.The spontaneous meiotic resumption of oocytes can be reversibly inhibited by incubation with cAMP-related inhibitors such as PDEs inhibitors or adenylate cyclase activators.In this study,we intend to test the effect of several inhibitors on mouse oocyte maturation arrest.The toxity of these inhibitors was also investigated.Inhibitors used in this study include PDEs inhibitors milrinone(Mil)and 3-isobutyl-1-methylxanthine(IBMX),adenylate cyclase activators Forskolin and dibutyryl cAMP(dbcAMP).All these inhibitors were used alone or in combination to find the best approach for mouse GV oocytes arrest.Our results indicated that when these inhibitors were used alone,the lowest dose for effective inhibition was:Mil:3.0 ?M,IBMX:30.0 ?M,Forskolin:450.0 ?M and dbcAMP:150.0 ?M.To figure out whether there would be better recipes when these regents were used in combination,different regents with different dose in combination were tested.The results showed that 50.0 ?M Forskolin + 0.3 ?M Mil,50.0 ?M Forskolin + 10.0 ?M IBMX,50.0 ?M dbcAMP +0.3 ?M Mil or 50.0 ?M dbcAMP + 10.0 ?M IBMX could inhibit oocytes at GV stage in a more efficient way.Further,when the group without any inhibitors was used as control,we found that althrough there was difference in terms of the GVBD progression when different inhibitors were used,the final ratio of oocytes completed GVBD was comparable in all nine groups.Next,we tested the effect of the inhibitors on first Polar Body(PB1)extrusion.The results showed that inhibitors of 30.0 ?M IBMX,50.0 ?M forskolin+ 0.3 ?M Mil or 50.0 ?M dbcAMP + 10.0 ?M IBMX had no influence on the progression and ratio of PB1 extrusion when compared with control group.In addition,inhibitors of 150.0 ?M dbcAMP,50.0 ?M Forskolin + 10.0 ?M IBMX or 50.0 ?M dbcAMP + 0.3 ?M Mil influenced the progression but not the ratio of PB1 extrusion when compared with control group.The PB1 size was also compared between control group and inhibition groups.The results showed that inhibitors of 50.0 ?M Forskolin + 0.3 ?M Mil,50.0?M Forskolin + 10.0 ?M IBMX or 50.0 ?M dbcAMP + 10.0 ?M IBMX induced relative smaller PB1,which was comparable with control group.In order to investigate the toxic effect of the upper inhibitors,oocyte early apoptotic signal,DNA damage as well as mitochondria distribution were tested before or after inhibitors treatment.In terms of oocyte early apoptotic signal,five kinds of inhibitors were found had not significant difference when compared with control group,including 30.0?M IBMX,150.0 pM dbcAMP,50.0 ?M Forskolin +0.3 pM Mil,50.0 ?M Forskolin + 10.0 ?M IBMX or 50.0 ?M dbcAMP + 10.0 ?M IBMX.In terms of DNA damage,three kinds of inhibitors were found had not significant difference when compared with control group,including 50.0 ?M Forskolin + 0.3 ?M Mil,50.0 ?M Forskolin + 10.0 ?M IBMX or 50.0 ?M dbcAMP+ 10.0 ?M IBMX.In terms of mitochondria distribution,only inhibitor of 50.0 ?M dbcAMP + 10.0?M IBMX had not significant difference when compared with control group.In sum,we concluded that the GV stage oocyte arrest effect can be improved if the combined inhibitors were used.And these combinations could minimize the inhibitors concentrations,which resulted in less toxity on oocytes maturation.Our results provided an improved recipe,50.0 pM dbcAMP + 10.0 ?M IBMX,for inhibiting oocyte maturation,which benefits the future research on oocyte maturation. |
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